TRAIL MRNA Modified Adipose-derived Stem Cells In Targeting Therapy Of Melanoma

Backgrounds and ObjetivesMalignant melanoma accounts for 1% of the whole body cancers. So far, melanoma is one of the most difficult types of cancers to successfully treat as it disseminates early after the development of the initial lesions. Almost all of the melanoma patients with advanced stage diseases succumb to distant metastases in 6-10 months after initial diagnosis. And the major obstacle in melanoma treatment is the lack of tumor specificity. Thus, more efficient treatment strategies specifically targeting tumor tissue are urgently required. Mesenchymal stem cells may have tumor-oriented homing capacity and thus potential as an efficient vehicle in patient-tailored cancer therapy. Furthermore, tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a promising anticancer death ligand,which has a killing effect on tumor tissues. According to these two characteristics, we designed this study. We utilized adipose-derived stem cells (ADSCs) to harbor TRAIL cDNA to facilitate TRAIL expression through the synthesis of mRNA in vitro and mRNA transfection technique,then we tested its effects on A375 melanoma cells in vitro by methods of ELISA, Transwell, Western blot etc.This study includes three parts:1. Isolation, culture and identification of ADSCs.2. Synthesis and quality testing of TRAIL mRNA.3. The anticancer effects of TRAIL-ADSCs on melanoma cells in vitro and to study its possible mechanisms.Methods and Results1. To isolate ADSCs from human adipose tissue samples, and certify the ADSCs having the phenotype and characteristics of normal MSCs. Using S A-?-gal to detect the effect of continuous passage on ADSCs aging, we found that the ADSCs can pass more than 10 generations in vitro. This effect could enable ADSCs obtain ample requirements of cell therapy, which can accelerate the development of cell therapy.2. We obtained a plasmid carrying TRAIL ORF sequences by transformation of pcDNA3.3-TOPO plasmid. Then, we got in vitro synthesis of TRAIL mRNA by using TAIL PCR of TRAIL ORF with capping, tailing and base modification. When transfected ADSCs with TRAIL mRNA, we can detect the stronger expressions of TRAIL at the protein level, which showed that TRAIL mRNA has better stabilities and stronger activities.3. We succeeded in obtaining sTRAIL fragments from pORF-sTRAIL target gene plasmid, and reconstructed it into pcDNA3.3-TOPO plasmid. When transfected ADSCs with TRAIL mRNA, we found that it has no significant effects on phenotype and cell activity of ADSCs. And we also found TRAIL-ADSCs could induce melanoma cells death after interactions of TRAIL-ADSCs and A375 melanoma cells, the mechanism of which may through activating caspase-3, caspase-8, caspase-9 and caspase-4 of apoptosis signaling pathways.ConclusionsTRAIL mRNA synthesized in vitro has better activities. When transfected ADSCs with TRAIL mRNA, TRAIL-ADSCs can effectively induce apoptosis of melanoma cells.